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- PDB-9f2k: Myo-inositol-1-phosphate synthase from Thermochaetoides thermophi... -

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Basic information

Entry
Database: PDB / ID: 9f2k
TitleMyo-inositol-1-phosphate synthase from Thermochaetoides thermophila in complex with NAD
Componentsinositol-3-phosphate synthase
KeywordsISOMERASE / inositol metabolism / endogenous / conformational selection
Function / homology
Function and homology information


inositol-3-phosphate synthase / inositol-3-phosphate synthase activity / inositol biosynthetic process / phospholipid biosynthetic process
Similarity search - Function
Myo-inositol-1-phosphate synthase / Myo-inositol-1-phosphate synthase / Myo-inositol-1-phosphate synthase, GAPDH-like / Myo-inositol-1-phosphate synthase / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / inositol-3-phosphate synthase
Similarity search - Component
Biological speciesThermochaetoides thermophila DSM 1495 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.48 Å
AuthorsTraeger, T.K. / Kyrilis, F.L. / Hamdi, F. / Kastritis, P.L.
Funding supportEuropean Union, Germany, 4items
OrganizationGrant numberCountry
European Union (EU)101086665European Union
German Federal Ministry for Education and Research03Z22HN23, 03Z22HI2 and 03COV04 Germany
European Regional Development FundZS/2016/04/78115European Union
German Research Foundation (DFG)391498659 Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Disorder-to-order active site capping regulates the rate-limiting step of the inositol pathway.
Authors: Toni K Träger / Fotis L Kyrilis / Farzad Hamdi / Christian Tüting / Marie Alfes / Tommy Hofmann / Carla Schmidt / Panagiotis L Kastritis /
Abstract: Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol ...Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus . By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications.
History
DepositionApr 23, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: inositol-3-phosphate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)57,3992
Polymers56,7351
Non-polymers6631
Water00
1
A: inositol-3-phosphate synthase
hetero molecules

A: inositol-3-phosphate synthase
hetero molecules

A: inositol-3-phosphate synthase
hetero molecules

A: inositol-3-phosphate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)229,5968
Polymers226,9424
Non-polymers2,6544
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation3

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Components

#1: Protein inositol-3-phosphate synthase


Mass: 56735.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Thermochaetoides thermophila DSM 1495 (fungus)
References: UniProt: G0SDP4, inositol-3-phosphate synthase
#2: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Native homotetramer of the Myo-inositol-1-phosphate synthase
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.24 MDa / Experimental value: NO
Source (natural)Organism: Thermochaetoides thermophila DSM 1495 (fungus)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
1100 mMHEPES1
295 mMNaCl1
35 mMKCl1
41 mMMgCl21
50.5 mMEDTA1
65 %Glycerol1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: 6 s blot time with a blot force of -1

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 240000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER / Temperature (max): 103.15 K / Temperature (min): 77.15 K
Image recordingElectron dose: 28 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 6261
Image scansWidth: 4000 / Height: 4000

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Processing

EM software
IDNameVersionCategoryDetailsFitting-ID
1cryoSPARC3.3.2particle selectionTemplate Picking
3EPU2.6image acquisitionData Collection
5cryoSPARC3.3.2CTF correctionPatch CTF Estimation
8UCSF ChimeraX1.6.1model fitting1
12PHENIX1.20.1-4487model refinement1
13UCSF ChimeraX1.6.1model fitting2
14Coot0.9.8.1model refinement2
15cryoSPARC3.3.2initial Euler assignmentAb-Initio Reconstruction
17cryoSPARC3.3.2final Euler assignmentNon uniform Refinement
19cryoSPARC3.3.2classification3D classification
21cryoSPARC4.4.13D reconstructionLocal refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 309043
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 255354 / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
IDProtocolSpace
1FLEXIBLE FITREAL
2FLEXIBLE FITREAL
Atomic model building
ID 3D fitting-IDDetailsSource nameType
11Local InstallationAlphaFoldin silico model
22Local InstallationAlphaFoldin silico model

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